Polymethine dyes, also known as cyanines, conform to the generalised formula:
Xxe2x80x94(CR)nxe2x80x94X1
in which n is an odd positive integer and (n+3) xcfx80 electrons are distributed over the polymethine chain and the terminal atoms X and X1; R, attached to the methine carbon C, is hydrogen or a radical. In the large majority of dyes X and X1 are the nitrogen atoms in a heterocyclic ring, but dyes are known in which one or both of the groups art non-cyclic or carbocyclic. The xe2x80x94CRxe2x80x94 groups can be replaced by one ore more aza (xe2x80x94Nxe2x95x90) links.
Comprehensive reviews regarding polymethine dyes have been by written by L. G. S. Brooker, xe2x80x9cThe Theory of the Photographic Processxe2x80x9d Mees Ed., Macmillan, New York, (1942), p. 987 and (1966), p. 198; Frances M. Hamer, in xe2x80x9cThe Chemistry of Heterocyclic Compoundsxe2x80x9d, Vol 18, xe2x80x9cThe Cyanine Dyes and Related Compoundsxe2x80x9d, Weissberger, Ed, Wiley Interscience, New York, (1964); G. E. Ficken, xe2x80x9cThe Chemistry of Synthetic Dyesxe2x80x9d, Vol 4, K. Venkataraman Ed., Academic Press, New York, (1971), p.211; A. I. Kiprianov, Usp. Khim., 29, 1336, (1960), 35, 361 (1966), 40, 594 (1971); D. W. Heseltine, xe2x80x9cThe Theory of the Photographic Processxe2x80x9d,4th edition, James Ed., Macmillan, New York, (1977), chapter 8, xe2x80x9cSensitising and Desensitising Dyesxe2x80x9d; S. Daehne, Phot. Sci. Eng., 12, 219 (1979); D. J. Fry, xe2x80x9cRodd""s Chemistry of Carbon Compoundsxe2x80x9d, xe2x80x9cCyanine Dyes and Related Compoundsxe2x80x9d, Vol. IVb, chapter 15, p.369 Elsevier, Amsterdam, (1977); Supplement to Vol. IVb, 2nd Edition (1985), p.267; H. Zollinger, xe2x80x9cColor Chemistryxe2x80x9d, VCH, Weinheim (1987), chapters 3 and 14; D. M. Sturmer, xe2x80x9cThe Chemistry of Heterocyclic Compoundsxe2x80x9d, xe2x80x9cSpecial Topics in Heterocyclic Chemistryxe2x80x9d, chapter VIII, xe2x80x9cSynthesis and Properties of Cyanine and Related Dyesxe2x80x9d, Weissberger Ed., Wiley, New York, (1977); xe2x80x9cThe Kirk-Othmer Encyclopaedia of Chemical Technologyxe2x80x9d Vol 7, p. 782, xe2x80x9cCyanine Dyesxe2x80x9d, Wiley, New-York, (1993).
For many years, polymethine dyes have been very useful as sensitisers in photography, especially in the red and near infrared regions of the spectrum. However, in more recent years, there has been an upsurge of new uses of these dyes in innovative technological areas, such as laser and electro-optic applications, optical recording media, medical, biological and diagnostic. These new applications of polymethine dyes place high demands on the degree of purity required, and the reproducibility of synthetic methods and purification steps is very important. These requirements are especially stringent for dyes designed to improve detection of ribonucleic acid (RNA), deoxyribonucleic acid (DNA) and of antigens in immunoassays. In these fields, the trend toward an increasing miniaturisation is accompanied by an increasing demand on sensitivity of the reporter molecules or labels. One way to increase the sensitivity of conventional fluorescence method is to use laser sources for the excitation. However, traditional fluorescent labels based on fluoresceins or rhodamins required expensive and/or bulky lasers. Moreover, their fluorescence occurs in the blue-green to green regions of the visible spectrum, where interference from the sample matrix is more likely to occur. Polymethine dyes do not suffer from these limitations. They can be efficiently excited by means of small, inexpensive solid state devices such as laser diodes or light emitting diodes, with extinction coefficients often several times higher than fluoresceins and rhodamines; they emit in the red and near-infrared regions of the spectrum, where non-specific fluorescence from the sample is low or lacking; another sources, Raman noise, becomes smaller with the inverse fourth power of wavelength (Imasaka, T., Yoshitake, A., and Ishibashi, N, xe2x80x9cSemiconductor Laser Fluorimetry in the Near-Infrared Regionxe2x80x9d, Anal. Chem., 56, 1077 (1984); Imasaka, T., and Ishibashi, N., xe2x80x9cDiode Lasers and practical trace Analysisxe2x80x9d, Anal. Chem., 62, 363 (1990); Matsuoka, M., Ed., xe2x80x9cInfrared Absorbing Dyesxe2x80x9d, Plenum Press, New York, (1990); J. Fabian, H. Nakazumi, M. Matsuoka, xe2x80x9cNear-Infrared Absorbing Dyesxe2x80x9d, Chem. Rev., 92, 1197, (1992); S. Daehne, U. Resch-Genger, O. S. Wolfbeis, xe2x80x9cNear-Infrared Dyes for High Technology Applicationsxe2x80x9d, Kluwer Academic Publishers, Dordrecht (1997).
To be useful as a label, a dye has to be provided with a suitable side chain containing a functional group. While the main part of the dye structure is generally known from previous applications, the introduction of a functional group into the structure for the purpose of conjugation, or binding to another molecule, represents the innovative step in the inventions concerning the use of the dye as a labelling reagent. In general, only one such functionalised side arm is preferable, in order to avoid cross-linking or purification problems. With a few exceptions, limited to heptamethine dyes, the standard approach in the design of polymethine labelling reagents has been to attach the functionalised side arm to one of the heterocyclic nuclei of the dye, formula (a):
HET1xe2x80x94HET2xe2x80x94Z
See, for instance: J. S. Lindsey, P. A. Brown, and D. A. Siesel, xe2x80x9cVisible Light-Harvesting in Covalently-Linked Porphyrin-Cyanine Dyes, Tetrahedron, 45, 4845, (1989); R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, and A. S. Waggoner, xe2x80x9cCyanine Dye Labelling Reagents Containing Isothiocyanate Groupsxe2x80x9d, Cytometry, 10, 11 (1989); L. A. Ernst, R. K. Gupta, R. B. Mujumdar, and A. S. Waggoner, xe2x80x9cCyanine Dye Labelling Reagents for sulphydryl Groupsxe2x80x9d, Cytometry, 10, 3, (1989); P. L. Southwick P. L., L. A. Ernst, E. W. Tauriello, S. R. Parker, R. B. Mujumdar, S. R. Mujumdar, H. A. Clever, and A. S. Waggoner, xe2x80x9cCyanine Dye Labelling Reagents-Carboxymethylindocyanine Succinimidyl Estersxe2x80x9d, Cytometry 11, 418 (1990); R. B. Mujumdar, L. A. Ernst, Swati R. Mujumdar, C. J. Lewis, and A. S. Waggoner, xe2x80x9cCyanine Dye Labelling Reagents: Sulfoindocyanine Succinimidyl Estersxe2x80x9d, Bioconjugate Chemistry, 4, 105, (1993); A. J. G. Mank, E. J. Molenaar, H. Lingeman, C. Goojer, U. A. Th. Brinkman, and N. H. Velthorst, xe2x80x9cVisible Diode Laser Induced Fluorescence Detection in Liquid Chromatography after Precolumn Derivatisation of Thiolsxe2x80x9d, Anal. Chem., 65, 2197, (1993); H. Yu., J. Chao, D. Patek, S. R. Mujumdar, and A. S. Waggoner, xe2x80x9cCyanine dye dUTP analogs for enzymatic labelling of DNA Probesxe2x80x9d, Nucl. Acids Res 22, 3226, (1994); Z. Zho, J. Chao, H. Yu, and A. S. Waggoner, xe2x80x9cDirectly labelled DNA probes using fluorescent nucleotides with different length linkersxe2x80x9d, Nucl. Acids, Res, 22, 3226. A. J. G. Mank, H. T. C. van der Laan, , H. Lingeman, Cees Goojer, U. A. Th. Brinkman, and N. H. Velthorst, xe2x80x9cVisible Diode Laser-Induced Fluorescence Detection in Liquid Chromatography after Precolumn Derivatisation of Aminesxe2x80x9d, Anal. Chem., 67, 1742, (1995); S. R. Mujumdar, R. B. Mujumdar, C. M. Grant, and A. S. Waggoner, xe2x80x9cCyanine Labelling Reagents: sulfobenzoindocyanine succinimidyl estersxe2x80x9d, Bioconjugate Chemistry, 7, 356, (1996). Patent Literature: P. L. Southwick, and A. S. Waggoner, xe2x80x9cIntermediate for and Fluorescent Cyanine Dyes containing Carboxylic Acid Groupsxe2x80x9d, U.S. Pat. No. 4,981,977, Jan. 1, 1991; A. S. Waggoner, L. A. Ernst, and Mujumdar, R. B., xe2x80x9cMethod for Labelling and Detecting Materials Employing Arylsulfonate Cyanine Dyesxe2x80x9d, U.S. Pat. No. 5,268,486, Dec. 7., 1993; A. S. Waggoner, xe2x80x9cCyanine Dyes as Labelling Reagents for Detection of Biological and Other Materials by Luminescence Methodsxe2x80x9d, U.S. Pat. No. 5,627,027, May 6, 1996; A. S. Waggoner, and R. B. Mujumdar, xe2x80x9cRigidised Trimethine Cyanine Dyesxe2x80x9d, WO99/311181; G.-Y. Shen, T. S. Dobashi, xe2x80x9cCyanine Dye Activating Group with Improved Coupling Selectivityxe2x80x9d; T. S. G. M. Little, R. Raghavachari; N. Narayanan; H. L. Osterman, xe2x80x9cFluorescent Cyanine Dyesxe2x80x9d, U.S. Pat. No. 6,027,709, Feb. 22, 2000.
The general synthetic strategy necessary to prepare these labelling reagents is as follows. First, a quaternised nitrogen heterocycle HET1 is prepared. Then, this heterocyclic base is reacted with an electrophilic reagent such as PhNHxe2x80x94(CHxe2x95x90CH)nxe2x80x94CHxe2x95x90NHPh.HCl or ROxe2x80x94(CHxe2x95x90CH)nxe2x80x94CH(OR)2, where Ph is a phenyl ring and R a methyl or ethyl group, to obtain a so-called hemicyanine dye, HET1xe2x80x94(CHxe2x95x90CH)nNHPh or HET1xe2x80x94(CHxe2x95x90CH)nNAcPh, where Ac is the acetyl radical, or HET1xe2x80x94(CHxe2x95x90CH)nxe2x80x94OR. These intermediates are then reacted with a different quaternary nitrogen heterocycle, HET2. The functionalised side arm can be attached either to the first or to the second quaternised nitrogen heterocycle. The final result is an asymmetric polymethine labelling reagent, HET1xe2x80x94(CHxe2x95x90CH)nxe2x80x94HET2xe2x80x94Z.
Unfortunately, the hemicyanine intermediates are notoriously difficult to obtain in good yields and/or in a pure form (see, for example, F. M. Hamer, xe2x80x9cSome Unsymmetrical Pentamethincyanine Dyes and their Tetramethin Intermediatesxe2x80x9d, J. Chem. Soc., 32 (1949) and R. B. Mujumdar, L. A. Ernst, Swati R. Mujumdar, C. J. Lewis, and A. S. Waggoner, xe2x80x9cCyanine Dye Labelling Reagents: Sulfoindocyanine Succinimidyl Estersxe2x80x9d, Bioconjugate Chemistry, 4, 105, (1993); in particular, note that when Mank (Anal. Chem., 67, 1744) tried to synthesise an asymmetric dicarbocyanine label described in the previous reference he obtained a total yield of 18% of dicarbocyanines, from which the desired product was difficult to separate; therefore he devised an alternative approach based on 1,3,3-trimethoxypropene. Unfortunately, this chemical is no longer available commercially.
In order to avoid such difficulties, the present invention is based on an alternative approach to the design of polymethine dyes with a single functionalised side arm. This general approach is illustrated in formula (b), below: 
In this case, the functionalised side arm Z is attached to the centre of the dye molecule, resulting in a symmetric labelling reagent. It is immediately obvious from this scheme that only one type of heterocyclic base, HET, is necessary and that the dye can be synthesised in one step, from HET (2 equivalents) and an electrophilic reagent bearing the functionalised side arm. The overall result is a much more convergent, more efficient synthesis of the required labels.
However, thus far, this approach has only found very limited application: the main example is in the synthesis of an isothiocyanate derivative of an heptamethine dye: L. Strekowski, M. Lipowska, and G. Patonay, xe2x80x9cFacile Derivatisation of Heptamethine Cyanine Dyesxe2x80x9d, Synth. Comm., 22(17), 2593-2598 (1992); L. Strekowski, M. Lipowska, and G. Patonay, xe2x80x9cSubstitution Reactions of a Nucleofugal Group in Heptamethine Cyanine Dyes. Synthesis of an Isothiocyanate Derivative for Labelling of Proteins with a Near-Infrared Chromophorexe2x80x9d J. Org. Chem. 57, 4578, (1992); N. Narayan, and G. Patonay, xe2x80x9cA New Method for the Synthesis of Heptamethine Cyanine Dyesxe2x80x9d, J. Org. Chem. 60, 2391-2395, (1995). No example was given in the case of pentamethine labelling reagents and only one example for their trimethine analogues (Compound IX in WO99/311181).
The present inventors succeeded in synthesising symmetric monofunctionalised polymethine dyes as shown in formula (b) having a wide variety of functional groups other than isothiocyanates and also having different functionalised arm chain lengths, which can be used for the labelling of a wide range of analytically and diagnostically useful biomolecules.
Examples of such analytically and diagnostically useful biomolecules include, but are not limited to, nucleotides and nucleosides, oligonucleotides, vitamins, proteins such as for example antibodies, antigens, streptavidin, and the like.
The dyes of the present invention are obtained by reacting 2 moles of quaternised nitrogen heterocycle base, HET, with suitable electrophilic reagents, such as diphenylformamidines or trialkylorthoformates and their vinilogs. The functionalised side arm can either be attached to the electrophilic reagent in a previous step, or after the formation of the polymethine dye structure.
The quaternised heterocyclic nuclei, HET, are commercially available, or can be synthesised by known methods from commercially available precursors. For example, the following heterocycle bases are all commercially available: 2,3,3-trimethyl-3-H-indole, 1,1,2-trimethyl-1-H-benz(e)indole, 2-methylbenzothiazole, 2-methylbenzoxazole,2-methylnaphth[1,2-d]thiazole, 2-methylnaphth[1,2-d]oxazole, 2-methyl-naphth[2,1-d]oxazole.
Other heterocyclic nuclei can be synthesised by known methods. For example, sulphonated indoles can be made from the corresponding aminosulphonic acids: these compounds are first converted to the corresponding hydrazinosulphonic acids by diazotisation followed by reduction with tin (II) chloride or other reducing agents, especially SO2 and sulphites; in the next step the hydrazine intermediates were condensed with 2-methylbutanone to yield the corresponding indoles, and then alkylated at the nitrogen with alkyl halides or sultones. Sulphonated benzo- and naphthoxazoles were obtained from the corresponding aminophenol and aminonaphtolsulphonic acids by condensation with acetic anhydride. A similar approach was used to prepare sulphonated benzo- and naphthothiazoles. 2-methyl-naphth[2,1-d]thiazole, was similarly prepared by condensation with acetic anhydride.
N, xcex1-alkylene cyclammonium salts of 3,3-dimethyl-3-H-indole, 1,1,2-dimethyl-1-H-benz(e)indole and their sulphonated analogues, were obtained by following the methods disclosed by L. L. Lincoln and D. W. Heseltine in xe2x80x9cMerocyanine Sensitisers for Silver Halidexe2x80x9d, U.S. Pat. No. 3,282,932 (1966), L. L. Lincoln and L. G. S. Brooker in xe2x80x9cPhotographic Sensitising Dyes of the Merocyanine And Styryl Types in Silver Halide Photographic Emulsionsxe2x80x9d, U.S. Pat. No. 3,397,981 (1968); G. L. Oliver in xe2x80x9cPhotographic Silver Halide Emulsions Containing N, xcex1-alkylene Bridged Merocyanine Sensitising Dyesxe2x80x9d, U.S. Pat. No. 3,403,026 (1968); G. L. Oliver, in xe2x80x9cSilver Halide Emulsions Containing N, xcex1-alkylene Bridged Indocyanine Sensitising Dyesxe2x80x9d, U.S. Pat. No. 3,408,195 (1968). N, xcex1-alkylene cyclammonium salts of benzothiazole, benzoxazole, naphth[1,2-d]thiazole, naphth[2,1-d]oxazole, naphth[1,2-d]oxazole, naphth[2,1-d]oxazole, and their sulphonated analogs were prepared according to the methods described by F. DS. Babichev, and N. Ya Derkach, Ukr. Khim. Zh., 22, 208 (1956); F. S. Babichev, and Neplyuev, xe2x80x9cBenzothiazolylalkyl Carboxylic Acids and their Derivatives-IV-Benzothiazolylalkyl Carbinolsxe2x80x9d, Zh. Obsch. Khim., 32, 857 (1962);); F. S. Babichev, and Neplyuev, xe2x80x9cBenzothiazolylalkyl Carboxylic Acids and their Derivativesxe2x80x94V-2,3-Polymethylenebenzothiazolium Saltsxe2x80x9d, Zh. Obsch. Khim., 32, 860(1962); F. S. Babichev xe2x80x9cCondensation of o-Aminobenzenethiol with Lactonesxe2x80x9d, Zh. Obsch. Khim., 33, 3016, (1963).
Methods for the synthesis of 1-Alkyl-2-methyl-benzo[c,d]indole nuclei, were provided by Ya. B. Shteinberg, in the article xe2x80x9cBenzo[c,d]indocyaninesxe2x80x9d, Khim. Geterotsik. Soedin. 3, 340 (1973) and by F. A. Mikhailenko, N. P. Vasilenko, A. D. Kachkovskii and Yu. I. Rozhinskii in xe2x80x9cEffect of Polar Substituents and the Length of the Polymethine Chain on the Color of Cyanine Dyes of the Benzo[c,d]indole Seriesxe2x80x9d, Zh. Org. Khim., 18, 435 (1982).
In the assembly of the dyes of this invention, two identical molecules of the above described quaternised nitrogen heterocycles are condensed with one molecule of electrophilic intermediates, which provide the bridging methine carbon atoms. For example, N,N-diphenylformamidines or trialkylorthoformates each add one methine carbon atom to the polymethine chain, giving rise to trimethincyanines or carbocyanines; malonaldehyde dianils or trialkoxypropenes contribute three methines to pentamethincyanines, or dicarbocyanines; and glutaconaldehyde dianils introduce five methines, to produce heptamethincyanines or tricarbocyanines, and so on. In one aspect of this invention, the functionalised side arm needed for linking the dye to another molecule, can be inserted into the middle, or meso (xcexc), position of the polymethine chain either before, or after the synthesis of the cyanine skeleton. Especially useful for this purpose are certain electrophilic reagents bearing a halogen atoms at the meso position, or cyanine dyes with halogens attached at this position. For example, meso-chloro- or bromomalonaldehyde dianils can be made from mucochloric or mucobromic acids by treatment with ethanol and aniline hydrochloride according to the directions given in Dieckman and Platz, Berichte 4639 (1904). These compounds can be used to synthesise the corresponding mesochloro, or bromodicarbocyanines. Similarly, the Vilsmeier-Haack-Arnold reaction can be used to prepare cyclic analogs of glutaconaldehyde bearing a halogen atom in the meso position: this structure is then incorporated into the corresponding cyanine dye. For example, S. M. Makin, L. I. Boiko and O. A. Shavrygina describe the synthesis of (5-phenylamino-2,4-trimethylene-3-chloro-2,4-pentadienylidene)phenylammonium chloride from cyclohexanone and the complex formed by mixing N,N-dimethylformamide and phosphorus oxychloride, xe2x80x9cAminoformylation of Unsaturated Aldehydes, 2-Alkoxyaldehydes and their Acetals, and Ketones of the Alicyclic Seriesxe2x80x9d, Zh. Org. Khim., 13, 1189 (1977). In our hands, the corresponding reaction with cyclopentanone only produced monoaminoformylated derivatives. The corresponding meso-bromo derivatives can be obtained by using phosphorus bromide in place of the oxychloride as shown by A. I. Ponogaev and S. M. Makin in xe2x80x9cMeso-bromo-substituted Tricarbocyanines with Cyclic Fragments in the Conjugation Chainxe2x80x9d, Zh. Org. Khim. 17, 167 (1980). These methods were applied successfully to 2-indanone by G. A. Reynolds and K. H. Drexhage, in xe2x80x9cStable Heptamethine Pyrylium Dyes that Absorb in the Infraredxe2x80x9d, J. Org. Chem, 42, 885 (1977) and by G. M. Sosnovskii, A. P. Lugovskii, and I. G. Tishchenko in xe2x80x9cSynthesis of Meso-substituted Tricarbocyanine Dyes with an Ortho-phenylene Bridge in the Chromophorexe2x80x9d, Zh. Org. Khim. 19, 2143 (1983). Also, in the later article, methods are described for the introduction of a phenyl substituent in the meso-position or the substituted cyclic glutaconaldehyde intermediates: the cycloalkanones are reacted with PhMgBr or PhLi yielding the corresponding alcohols which can be easily dehydrated; these intermediates are subjected to a two-step aminoformylation, first with dimethylformamide dimethyl acetal and then with the DMF-POCl3 complex. Functionalised side arms can be introduced in the para position of the phenyl substituent, by masking the functionality with appropriate protective groups, i.e. dioxanes or dioxolanes for aldehydes, oxazolines for carboxylic groups and tetrahydropyranes for alcohols, as described in the book by T. W. Greene and P. G. M. Wuts, xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, John Wiley and Sons, New York, N.Y. (1991).
Trimethincyanines with a meso chloro groups are best made as disclosed by G. L. Oliver in xe2x80x9cPhotographic Emulsions and xcexc-chlorocarbocyanine Dyesxe2x80x9d, U.S. Pat. No. 3,656,960 (1962).
In trimethincyanines and heptamethine cyanines the meso halogen is easily displaceable by more nucleophilic atoms such as O, S, Se, and N. This provides a convenient route for the introduction of a great number of functionalised side arms, by using reagents bearing a functional group at their distal end. As was shown above, this method found some very limited application by Patonay and his group, in the preparation of heptamethincyanines with an isothiocyanate reactive group at the distal end of a thiophenyl meso substituent. This reagent has only very limited utility, mostly for cell or protein labelling, while it is totally unsuitable for the labelling of small molecules such as nucleotides. It is the purpose of this invention to provide better compounds by this route for applications where it is important to limit the perturbation caused to much the labelled molecule.
The above method could not be extended to pentamethincyanines, where the meso halogen not easily displaceable by nucleophiles. This effect is due to the alternation of charge density in the meso methine carbon in the series tri-, penta- and heptamethine dyes. In other words, the halogen-carbon bond in the meso position of pentamethincyanines is similar to that found in vinyl or aromatic halides. On this basis, it is possible to exploit the methods developed for the creation of carbon-carbon bonds from sp2 halides and unsaturated hydrocarbons with the help of palladium catalysts, as described by R. F. Heck in xe2x80x9cPalladium Reagents in Organic Synthesisxe2x80x9d, Academic Press, New York, N.Y., 1985; J. Tsuji, in xe2x80x9cPalladium Reagents and Catalystsxe2x80x9d, John Wiley and Sons, New York, N.Y., 1995; and by J.-L. Malleron, J.-C-Fiaud, and J.-Y. Legros, in xe2x80x9cHandbook of Palladium-Catalysed Organic Reactionsxe2x80x9d, Academic Press, New York, N.Y., 1997. Especially useful in our context is the reaction between sp2 halides and alkynes bearing a functional groups. These reactions are tolerant of many functionalities, occur under mild condition and in high yields. Similar methods proved successful also in the case of trimethine and heptamethincyanines, where the meso halogen is much more labile.
Other methods were developed for pentamethincyanines. In some cases, it is possible to synthesise pentamethine cyanines bearing useful functional groups in the meso position, for example an ester group, as disclosed by F. P. Doyle, in xe2x80x9cImprovements in or Relating to the Production of Cyanine Dyestuffs and to the Sensitising of Photographic Emulsionsxe2x80x9d, G.B. Patent No. 640,127 (1950). The Vilsmeyer-Haack-Arnold reaction is also very useful for the preparation of meso-substituted malondialdehydes needed for the preparation of the corresponding meso-substituted pentamethincyanines, C. M. Marson and P. R. Giles, xe2x80x9cSynthesis Using Vilsmeier Reagentsxe2x80x9d, CRC Press, Boca Raton, Fla., 1994. For example by treating substituted acetic acids, Rxe2x80x94COOH, where R is aryl, chloro, ethoxycarbonyl, with an excess of the Vilsmeier reagent N,N.dimethylformamide-POCl3 results in substituted malonaldialdehyde synthetic equivalents, CH3N+xe2x95x90Cxe2x80x94CRxe2x95x90CHxe2x80x94N(CH3)2.
The subject-matter of the present invention is therefore constituted by symmetric cyanine labelling dyes having the general formula (1): 
wherein:
X is selected from the group consisting of O, S and C(CH3)2;
W represents non-metal atoms required to form a benzo-condensed or a naphto-condensed ring;
R1 is selected from the group consisting of (CH2)nCH3, (CH2)nSO3xe2x88x92 and (CH2)nSO3H, wherein n is an integer selected from 0 to 6 when R1 is (CH2)nCH3, and n is an integer selected from 3 to 6 when R1 is (CH2)nSO3xe2x88x92 or (CH2)nSO3H;
R2 and R3 are independently selected from the group consisting of H, a sulphonic moiety and a sulphonate moiety;
Q is selected from the group consisting of: 
wherein q is 0 or 1 and D is selected from the group consisting of: 
wherein A is O or S and G is, or contains a N, O or S nucleophile moiety or is, or contains a moiety capable of reacting with N, O or S nucleophiles.
It is understood that the case where q=0 refers to a ring having 5 carbon atoms.
Preferably, the N, O or S nucleophile moiety is selected from the group consisting of: (CH2)mOH, (CH2)mNH2, (CH2)mSH, (CH2)mY(CH2)pOH, (CH2)mY(CH2)pNH2, and (CH2)mY(CH2)pSH.
Preferably, the moiety capable of reacting with N, O or S nucleophiles is selected from the group consisting of: (CH2)mCOOH, (CH2)mglycidyl, (CH2)mmaleimide, (CH2)mCOxe2x80x94NHS, (CH2)mCO-imidazole, (CH2)mSO2CHxe2x95x90CH2, (CH2)mCONHNH2, (CH2)mCHO, (CH2)mY(CH2)pCOOH, (CH2)mY(CH2)pglycidyl, (CH2)mY(CH2)pmaleimide, (CH2)mY(CH2)pCOxe2x80x94NHS, (CH2)mY(CH2)pCO-imidazole, CH2(CH2)mOxe2x80x94PAM, (CH2)mY(CH2)pSO2CHxe2x95x90CH2, (CH2)mY(CH2)pCONHNH2, (CH2)mY(CH2)pCHO, and (CH2)mY(CH2)pOxe2x80x94PAM, wherein 
In the above formulae Y is selected from the group consisting of xe2x80x94NHxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94Oxe2x80x94 and xe2x80x94Sxe2x80x94, m is an integer selected from 0 to 6 and p is an integer selected from 1 to 6.
In the above illustrated symmetric cyanines, it is preferred that at least one of the moieties R1 to R3 is, or contains a sulfonic or a sulphonate moiety.
In a preferred embodiment of the symmetric cyanines of the invention, X is C(CH3)2 and one of the moieties R2 and R3 is a sulphonic moiety or a sulphonate moiety; according to this embodiment of the invention, R1 is more preferably (CH2)nSO3xe2x88x92 or (CH2)nSO3H.
In another preferred embodiment of the symmetric cyanines of the invention, X is S and R1 is (CH2)nSO3xe2x88x92 or (CH2)nSO3H.
Also salts of the above illustrated symmetric cyanines are within the scope of the present invention. Examples of such salts include, but are not limited to, chloride, iodide and bromide salts; sodium, potassium and magnesium salts.
Also the valence tautomers of the symmetric cyanines of formula (1) are included within the scope of the invention, wherein the valence tautomerism is intended to mean the shifting of the conjugated bonds in the polymethine chain. Examples of symmetric cyanines of the present invention are compounds of formula (1) in which G, R1, R2, R3 have the meanings illustrated in table 1 below.
Each sulphonic moiety in the above table may be replaced by a corresponding sulphonate moiety.
Preferred symmetric cyanines according to the present invention are represented by any of the formulae (2a) to (2l): 
wherein R1, R2, R3, X, q and D have the meanings indicated in respect of formula (1).
The following examples are simply meant to further illustrate specific applications of the present invention and are not intended to be construed as defining or limiting the scope of the invention.